Screening cloned prokaryotic or eukaryotic hosts for protein expression and function is of great importance in the field of biotechnology. However, this technology requires evaluating each individual clone for protein expression and function. Often it is necessary to examine large numbers of cloned hosts to determine which expression system is most efficient or which protein has the characteristic function that is most desirable.
Mutagenesis is a powerful method for evaluating protein expression and function. Mutagenesis involves modifying one or more bases in a nucleotide sequence to express the protein of interest. However, because there are no reliable methods to predict the effect of modified nucleotide sequences, large numbers of point mutants and mutant proteins need to be individually evaluated.
In the past, screening individual mutants for protein expression and function was extremely time consuming and very labor intensive because individual samples needed to be evaluated. It was also very difficult to determine which point mutant expressed the protein of interest.
Proliferating cell nuclear antigen (PCNA) exemplifies this difficulty. Previously, the evaluation of PCNA point mutants was performed by testing individual clones for their effects in vitro on purified DNA polymerase xcex4(pol xcex4) using conventional protein identification and purification techniques of mutant PCNA molecules. This approach while effective, is very labor intensive. (See, for example, Refs. 9-11).
The isolation and purification of cloned proteins has been greatly facilitated by the use of affinity tags. A widely used affinity tag is a histidine (his) tag. These tags typically contain a string of four to ten consecutive histidine residues genetically engineered to be at either the amino- or carboxyl-terminus of recombinant proteins. Because his-tagged proteins bind to chelated metals attached to solid supports, they can easily be isolated by column chromatography with chelated metals attached to resins or beads as the solid support. Such chromatographic methods are referred to as Immobilized Metal Ion Affinity Chromatography (IMAC).
The use of IMAC for isolating proteins was first disclosed in Porath et al., (Ref. 31), wherein a resin was derivatized with iminodiacetic acid (IDA) and metal ions were chelated to the IDA-derivatized resin for immobilizing proteins. Columns with nickel-agarose or metal containing resin are typically used for isolating his-tagged proteins. The his-tagged protein binds to the matrix by interacting with the metal ions and is then eluted with a solution of imidazole, competing metal ions or salt. Unfortunately, the use of columns for detecting his-tagged proteins is very labor intensive and not amenable to high throughput screening.
An alternate method for identifying his-tagged proteins is disclosed in the QIAGEN product guide 1997. The QIAGEN method is a two dimensional method for detecting his-tagged proteins that uses Ni-nitrilotriacetic acid (Ni-NTA) attached to a column matrix to bind his-tagged proteins.
Similarly, a method for identifying his-tagged proteins on micro titter plates is disclosed in Paborsky et al., (Ref. 29). This method utilizes maleic anhydride-activated polystyrene microtiter plates coupled with N,N-bis[carboxymethyl]lysine, a derivative of nitrilotriacetic acid (NTA), to immobilized histidine-containing proteins. Paborsky also discloses a method for quantifying expression levels of the immobilized protein. These methods, although useful, are very labor intensive and not amenable to high throughput screening.
Based on the foregoing, there is still a great need for alternate methods and compositions for screening large numbers of cloned mutants for both protein expression and function. Methods and compositions that allow two-dimensional high throughput screening would be of particular value for designing proteins for pharmaceutical and industrial uses.
These and other objectives are achieved by the present invention, which in one embodiment provides a modified cellulose for detecting a protein of interest, comprising metal charged iminodiacetic acid cellulose.
In another embodiment, the present invention provides a process for preparing modified cellulose for his-tag protein binding, comprising reacting cellulose epoxide with iminodiacetic acid to form iminodiacetic acid-cellulose; and incubating the iminodiacetic acid-cellulose with a metal salt thereby preparing a metal charged iminodiacetic acid cellulose for binding his-tagged proteins.
In yet another embodiment, the present invention provides a method for screening a test sample to determine if the sample is his-tagged, comprising contacting metal charged iminodiacetic acid cellulose with the test sample; washing the iminodiacetic acid-cellulose; and detecting the his-tagged sample that remains immobilized on the metal charged iminodiacetic acid cellulose.
In one embodiment, the invention includes a method for determining protein expression, comprising introducing into cells a vector comprising a nucleic acid that encodes a protein of interest having a polyhistidine region; growing the cells that have the vector; preparing a replica of the cells that have the vector on a membrane support; expressing the protein of interest in the cells; lysing the cells on the membrane support in situ to release the protein of interest; transferring the protein of interest to a metal charged iminodiacetic acid cellulose; washing the metal charged iminodiacetic acid cellulose; and detecting the protein of interest that is immobilized on the metal charged iminodiacetic acid cellulose.
In another embodiment, the present invention includes a method for renaturing proteins, comprising denaturing a protein having a polyhistidine region; contacting the protein with metal charged iminodiacetic acid cellulose to transfer and bind the protein to the cellulose under denaturing conditions; renaturing the protein; and recovering or detecting the renatured protein.
A significant advantage of the metal charged iminodiacetic acid cellulose is that it allows for easy screening of a large number of proteins following mutagenesis. Accordingly, one skilled in the art can rapidly ascertain which mutants have desired functional activity or binding capacity.
The invention provides, for the first time, a two-dimensional screening system that is readily amenable to high throughput screening of cloned proteins. By maintaining a two-dimensional format, the present invention enables the processing of large numbers of samples concurrently. This is not possible with the three dimensional columns and gels available in the prior art.
These and other advantages of the present invention will be appreciated from the detailed description and examples set forth herein. The detailed description and examples enhance the understanding of the invention, but are not intended to limit the scope of the invention.